Aflatoxins are a group of highly toxic metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are one category of most powerful carcinogenic substances ever discovered. 20 kinds of aflatoxins have now been found, which mainly include aflatoxin B1 (AFB1), B2 (AFB2), AFG, and M1 (AFM1), etc. Among them, aflatoxin B1 is the most toxic and its toxicity is 10 folds of potassium cyanide and 68 folds of arsenic. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of AFB1. When the mammals ingest a feed contaminated by AFB1, AFB1 would be hydroxylated in vivo and secreted into milk. In general, after the animals ingest a food contaminated by AFB1, the discharge amount of aflatoxin M1 is 1%-3% of the intake amount of AFB1. A large number of researchers have conducted deep research on the toxicity and carcinogenicity of aflatoxin M1, and the research results motivate International Agency for Research on Cancer to change the carcinogenic rank of aflatoxin M1 from the category II carcinogenic substance to the category I carcinogenic substance. Aflatoxin M1 is stable in property and almost completely impossible to be destroyed even if it is subjected to pasteurization. Aflatoxin M1 is present in many dairy products. Since dairy products are the main source of infant foods, the problems about the aflatoxin M1 contamination have attracted worldwide attention and the amount of aflatoxin M1 is strictly limited in those dairy products. China belongs to heavily contaminated areas of aflatoxins, and therefore it is of important significance to intensify the detection especially the rapid detection of aflatoxin M1 in milk and dairy products to timely understand and grasp the health information about the milk and dairy products for ensuring the safety of food consumption in China.
The existing detection methods for aflatoxins include chemical analysis method, precision instrument analysis method and an immunological analysis method. Among them, the chemical analysis method is the most commonly used detection method for aflatoxins. The chemical analysis method does not need special instruments and equipment and can be carried out in ordinary laboratories, but it has the problems of large reagent consumption, tedious operation, severe interference by other components, poor accuracy, incapability of accurate quantification, great harm to experimenters and surrounding environment, and inapplicability to in-field rapid detection. The precision instrument analysis method includes fluorospectrophotometry and high performance liquid chromatography. The precision instrument analysis methods have high sensitivity and good accuracy, but the instruments therefor are expensive. Further, those methods require a high purification of aflatoxin samples, require tedious sample pretreatment procedures, are long time consumption, require high standard of experimental environment, and are difficult to realize rapid detection. Immunological analysis technology developed in recent years overcomes the disadvantages of the former two methods, has the advantages of strong specificity, high sensitivity, simple sample pretreatment, low cost, less contamination harm to experimenters and surrounding environment, applicability to in-field batch detection, etc., and has been applied in many fields such as food and medical treatment. Immunological analysis is based on the specificity between an antigen and an antibody and their reversible binding reactions. Using the antigen and the antibody as biochemical reactants to carry out qualitative and quantitative analysis on substances such as compounds, enzymes or proteins, has the advantages of good sensitivity and rapidness and simple operation. An anti-aflatoxin antibody must be prepared in advance in order to study and establish any immunological detection technique directed to aflatoxins.
Immunological analysis needs high-quality antibodies. With the development of antibody technology, recombinant antibodies are gradually applied in the aflatoxins detection field. Compared with traditional polyclonal antibodies and monoclonal antibodies, the recombinant antibodies have unique advantage in that recombinant antibodies are easily mass produced in protokaryotic expression system within a very short period of time with low production cost. Thus, recombinant antibodies have important application values for the low cost and large scale detection of aflatoxins, and can satisfy the growing demands for the production of aflatoxin antibodies. Nanobodies are variable region fragments of heavy chains of natural antibodies in an animal body of camelidae which are obtained by employing molecular biological means and can be specifically bound to antigens. Compared with traditional recombinant antibodies (such as single-chain antibodies), nanobodies have the advantages of small volume, good stability, tolerance to high temperature, tolerance to organic reagents, tolerance to acids and bases and the like. Currently, there is still no report related to aflatoxin M1 nanobodies.